Comparative proteomic analysis of wall-forming bodies and oocyst wall reveals the molecular basis underlying oocyst wall formation in Eimeria necatrix

Background The durable oocyst wall formed from the contents of wall-forming bodies (WFBs) protects Eimeria parasites from harsh conditions and enhances parasite transmission. Comprehending the contents of WFBs and proteins involved in oocyst wall formation is pivotal to understanding the mechanism of the oocyst wall formation and the search for novel targets to disrupt parasite transmission. Methods Total proteins extracted from WFBs and the oocyst wall of Eimeria necatrix were subjected to comparative proteomic analysis using tandem mass tag in conjunction with liquid chromatography tandem-mass spectrometry techniques. After functional clustering analysis of the identified proteins, three proteins, including E. necatrix disulfide isomerase (EnPDI), thioredoxin (EnTrx) and phosphoglycerate kinase (EnPGK), were selected for further study to confirm their potential roles in oocyst wall formation. Results A total of 3009 and 2973 proteins were identified from WFBs and the oocyst wall of E. necatrix, respectively. Among these proteins, 1102 were identified as differentially expressed proteins, of which 506 were upregulated and 596 downregulated in the oocyst wall compared to the WFBs. A total of 108 proteins, including compositional proteins of the oocyst wall, proteases, oxidoreductases, proteins involved in glycosylation, proteins involved in synthesis of the acid-fast lipid layer and proteins related to transport, were proposed to be involved in oocyst wall formation. The approximate molecular sizes of native EnPDI, EnTrx and EnPGK proteins were 55, 50 and 45 kDa, respectively. EnPDI was present in both type 1 and type 2 WFBs, EnTrx was present only in type 2 WFB2 and EnPGK was present only in type 1 WFBs, whereas all of them were localized to the outer layer of the oocyst wall, indicating that all of them participate in the formation of the oocyst wall. Conclusions To the best of our knowledge, this is the first report on the proteomes of WFBs and the oocyst wall of E. necatrix. The data obtained from this study form a basis for deciphering the molecular mechanisms underlying oocyst wall formation of Eimeria parasites. They also provide valuable resources for future studies on the development of novel therapeutic agents and vaccines aimed at combating coccidian transmission. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s13071-023-06076-6.


Background
Avian coccidiosis, a parasitic disease caused by one or more of seven species of the genus Eimeria, is one of the most widely reported diseases within the poultry industry worldwide [1,2].Eimeria necatrix is highly pathogenic and causes major lesions in the small intestine and substantial mortality, particularly in chickens older than 8 weeks raised on litter floors [1].Conventional coccidiosis control strategies rely heavily on chemoprophylaxis and, to a certain extent, live vaccines [3].Annually, the poultry industry spends about £7.7 to £13.0 billion (at 2016 prices) in only seven countries on prophylaxis, treatment and production losses due to avian coccidiosis [4].In this context, avian coccidiosis remains one of the major problems globally to date.
Eimeria parasites undergo complex life-cycles involving asexual proliferation followed by sexual development leading to the production of oocysts [1].Prior to excretion in the feces of the infected hose, the oocyst is encapsulated by a hard barrier, the oocyst wall, which protects the parasite from the harsh external environment.Once excreted from the host, the oocyst develops further (sporulation) and is passed onto the next host via the fecal-oral route [5].The oocyst wall is vital for parasite survival in the external environment [5,6].Therefore, a good understanding of the molecular basis of oocyst wall formation may be relevant to the development of novel vaccines and drugs for treating the debilitating diseases caused by Eimeria parasites, as well as those caused by the related cyst-forming parasites Toxoplasma gondii, Cryptosporidium spp.and Neospora caninum, among others.
A great deal of research has focused on elucidating the structure, biochemical composition and developmental biology of the oocyst wall of Eimeria spp.[5][6][7].This research has so far revealed that the bilayered oocyst wall is formed from the contents of two organelles, wall-forming bodies type 1 and 2 (WFB1, WFB2), located exclusively in the macrogametocytes [8].WFB1s and WFB2s are synthesized during the maturation of the macrogametes and appear to give rise to the outer and inner layers of the oocyst wall, respectively [6].Compositional analysis of E. tenella and E. maxima showed that their oocyst walls were primarily composed of protein (> 90% of wall) and only relatively small amounts of lipid and carbohydrate [5,6].However, only a small number of oocyst wall proteins have been identified and characterized to date, and these are mainly tyrosine-rich proteins ranging in size from 8 to 31 kDa [9].Studies have revealed that these tyrosine-rich proteins are all derived from precursor proteins stockpiled in WFBs of macrogametes.During oocyst wall formation, these precursor proteins are proteolytically processed into smaller tyrosine-rich proteins prior to protein-tyrosine cross-linking and hardening of the oocyst wall [5].However, the contents of WFBs and the identity of the proteins and enzymes involved in oocyst wall formation remain poorly understood.
In the present study, we employed tandem mass tag (TMT) peptide labeling coupled with the liquid chromatography tandem mass spectrometry (LC-MS/MS) quantitative proteomics technique to investigate the protein abundance of WFBs and the oocyst wall of E. necatrix.Previous studies had revealed that protein disulfide isomerase (PDI) and thioredoxin (Trx) are involved in disulfide oxidoreduction and contribute significantly to cyst wall formation in Giardia [10].Trxs and PDI, both members of the thioredoxin superfamily, share a common thioredoxin fold and play roles in disulfide oxidoreduction and/or isomerization [11].Additionally, phosphoglycerate kinase (PGK) is a bona fide cell wall protein of Candida albicans [12] and is crucial for cytoskeletal rearrangements in Schistosoma mansoni [13].In this context, we aimed to determine whether E. necatrix PDI (EnPDI), EnTrx and EnPGK are involved in the oocyst wall formation of E. necatrix.We also aimed to identify the proteins potentially involved in oocyst wall formation.The results of this study would provide important information on oocyst wall formation and also contribute to new targets for avian coccidiosis control.

Parasites and animals
The E. necatrix Yangzhou strain used in this study was originally isolated from a chicken that died of coccidiosis caused by E. necatrix in 2009 in Yangzhou, China, as confirmed by microscopic examination and sequence analysis of the internal transcribed spacer region of extracted genomic DNA.This strain has been maintained in our laboratory according to the method previously described [14].
One-day-old chickens were obtained from the Poultry Institute of the Chinese Academy of Agricultural Sciences (Yangzhou, Jiangsu, China).The chickens were housed in Eimeria-free isolation cages and provided with clean water and adequate feed without anticoccidial drugs.Chicken feces were collected and analyzed by salt flotation and light microscopy to confirm the absence of oocysts in the chickens 1 day before the experimental inoculations.Chickens between 4 and 5 weeks of age were used to prepare gametocytes (GAM) and oocysts of E. necatrix.Six-week-old specific-pathogen-free female BALB/c mice were purchased from Yangzhou University (Comparative Medicine Center) and maintained under specific-pathogen-free conditions.These mice were used to prepare the antibodies against recombinant proteins.
All animal care and procedures were conducted according to the guidelines for animal use in toxicology.The study protocol was approved by the Animal Care and Use Committee of the College of Veterinary Medicine, Yangzhou University.

GAM preparation and WFB isolation
Gametocytes and WFBs were obtained following the method described previously [15].Briefly, E. necatrix second-generation merozoites (MZ-2) were obtained from the small intestine of chickens 136 h after oral inoculation with 2.0 × 10 4 E. necatrix sporulated oocysts.Following the administration of halothane to induce anesthesia, the ceca of chickens were exposed and the base of the ceca tied off with a cotton ligature.Subsequently, approximately 1.8 × 10 8 MZ-2 in a volume of 1.5-2 ml were injected into the cecal lumen using a syringe with a 25-gauge needle.At 30 ± 0.5 h after injection with MZ-2, the chickens were sacrificed and the ceca removed.The mucosal tissues were obtained and digested with hyaluronidase in SAC (1 mM phenylmethanesulfonyl fluoride, 1 mg/ml bovine serum albumin [BSA], 170 mM NaCl, 10 mM Tris-HCl pH 7.0, 10 mM glucose and 5 mM CaCl 2 ) to isolate the GAM.The GAM were purified using Percoll (GE Healthcare, Uppsala, Sweden) density gradient centrifugation as follows.First, 2 ml of 50% Percoll/PBS (volume ratio 1:1) solution was placed into a sterile 15-ml tube, followed by the slow addition of 5 ml of 30% Percoll/GAM suspension (volume ratio 3:7).The tube was then centrifuged at 3000 g for 20 min, following which the supernatant was discarded, and the GAM washed twice with cold PBS by centrifugation.The purified GAM (1 × 10 8 cells) were then extracted with 0.1% saponin in Tris-NaCl-EDTA (TNE) buffer for 20 min at room temperature and centrifuged at 1000 g for 5 min.After washing with TNE buffer, the pellet was sonicated in an ice water bath and the lysates filtered through a 5-μm polymon mesh.The filtrate was then added to 5% sodium dodecyl sulfate, vortexed and centrifuged at 15,000 g for 10 min.The WFBs in the pellet were purified using a 1000-kDa cutoff Vivaspin 6 centrifugal filter (Sartorius Stedim Biotech, Aubagne, France), centrifuged at 4000 g for 20 min and concentrated three times.The purified WFBs were obtained from the concentrated solution by centrifugation at 15,000 g at 4 °C for 10 min.Finally, WFB1 and WFB2 were confirmed by immunofluorescence co-localization using anti-recombinant E. necatrix GAM protein 22 (anti-rEnGAM22) and anti-recombinant E. necatrix GAM protein 59 (anti-rEnGAM59) polyclonal antibody (pAb) [15].The purified WFBs were stored at 4 °C or frozen immediately in liquid nitrogen for future use.Three biological replicates were performed to collect WFBs for proteomic analysis.

Isolation and purification of the oocyst wall
Oocysts were isolated and purified according to the method reported previously [14].Briefly, the 4-to 5-week-old chickens were orally infected with approximately 2 × 10 4 sporulated oocysts of E. necatrix.The feces were collected between 6 and 9 days after infection to separate oocysts out using a saturated salt solution and saturated sucrose solution, respectively.After the purified oocytes had been sterilized by sodium hypochlorite treatment and washed with sterile water, they were used to prepare the oocyst wall according to the previously described method [16] with modifications.Briefly, approximately 4 × 10 7 purified unsporulated oocysts were disrupted by vigorous vortexing in 2 ml of PBS containing 1 mM PMSF, 1 mM EDTA and 1 mM DTT and 2 g of acid-washed glass beads (≤ 106 μm; Sigma-Aldrich, St. Louis, MO, USA) for 5 min until > 80% of them were ruptured.The resulting mixture was resuspended in 5 ml of 0.5 M sucrose and layered onto 3 ml of 1.1 M sucrose in a 15-ml centrifuge tube and centrifuged at 3000 g for 15 min.The pellet was washed 5 more times by resuspension in 10 vol of PBS and centrifugation at 10,000 g.The final pellet, consisting of purified oocyst wall, was then lash-frozen in liquid nitrogen.Three biological replicates were performed to collect the oocyst wall for proteomic analysis.

Pre-treatment and TMT labeling of protein
Samples of WFBs and the oocyst wall were resuspended in lysis buffer containing 8.0 M urea, 0.1% SDS, 1× Protease Inhibitor Cocktail tablets (Roche, Indianapolis, IN, USA) and 1 mM PMSF (Biyotime Institute of Biotechnology, Shanghai, China) [17,18].After incubation on ice for 30 min, the lysed samples were centrifuged, followed by concentration through a 3-kDa filter (Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-3 membrane; MilliporeSigma, Burlington, MA, USA), and the resulting supernatant was stored at − 80 °C [17,19].Protein concentration was determined using the Pierce BCA Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol.Efficient protein extraction was confirmed via electrophoresis, with equal amounts (20 μg) of protein loaded onto an SDS-polyacrylamide gel electrophoresis (PAGE) gel and subsequent staining of the products with Coomassie Brilliant Blue R-250.
To generate peptides, the protein extracted from each sample was digested with 2.5 μg of trypsin (Thermo Fisher Scientific).These resulting peptides were then labeled using the TMT kit (TMT 10 plex ™ Isobaric Label Reagent Set; Thermo Fisher Scientific).Specifically, peptides within each experimental group were labeled with distinct TMT labels: three biological replicates of the oocyst wall group were designated as TMT-126, TMT-127C and TMT-127N, while three biological replicates of the WFB group were labeled as TMT-128C, TMT-128N and TMT-131.

High-performance liquid chromatography fractionation and LC-MS/MS analysis
The TMT-labeled peptides were dissolved in solvent A buffer (98% double-distilled water, 2% acetonitrile, pH 10) and fractionated by high-pH reversed-phase fractionation chromatography utilizing an XBridge C18 column (5 μm, 250 × 4.6 mm; Waters Corporation, Milford, MA, USA).Fractions were collected every 1 min in 40 tubes, subsequently dehydrated and then amalgamated into 10 tubes to facilitate subsequent LC-MS/MS analysis.
LC-MS/MS analysis was performed on the U3000 Nano-Scale Liquid Chromatography System (Thermo Fisher Scientific) coupled with a Q-Exactive mass spectrometer (Thermo Fisher Scientific) via a nano-electrospray source.TMT-labeled peptides were loaded onto a 25-cm-long, 75-μm inner diameter fused silica analytical column packed with 2.0 μm Aqua C18beads (Thermo Fisher Scientific), using an autosampler at 5 μl/min.Elution utilized a gradient of buffer A (0.1% formic acid in water) and buffer B (0.1% formic acid in acetonitrile) at a flow rate of 300 nl/min for 43.5 min.Ion signals were acquired in data-dependent mode, with a full scan resolution of 70,000 and a scan range of m/z 350-1600.The resulting MS/MS data were saved as raw files using Xcalibur software version 2.2 (Thermo Fisher Scientific).TMT-proteomic analysis was conducted by CapitalBio Corporation, Beijing, China.The proteomics data generated by mass spectrometry have been submitted to the ProteomeXchange Consortium through the PRIDE partner repository under the dataset identifier PXD042839 and can be accessed at https:// www.ebi.ac.uk/ pride/ archi ve.

Protein identification and quantitative analysis
The raw MS data were analyzed using Proteome Discoverer 2.3 software, which was used for database retrieval, peptide mapping and protein quantitation.The MS/MS spectra were searched against five reference databases, namely those of E. necatrix (https:// www.ncbi.nlm.nih.gov/ assem bly/ GCF_ 00049 9385.1/),E. tenella (https:// www.ncbi.nlm.nih.gov/ assem bly/ GCF_ 00049 9545.2/),E. maxima (https:// www.ncbi.nlm.nih.gov/ assem bly/ GCF_ 00049 9605.1/),T. gondii (https:// www.ncbi.nlm.nih.gov/ assem bly/ GCF_ 00000 6565.2/) and Plasmodium falciparum (https:// www.ncbi.nlm.nih.gov/ assem bly/ GCF_ 00000 2765.4/).To control the false discovery rate (FDR) at the protein and peptide levels, we applied a fusiondecoy database search strategy with a threshold of ≤ 1.0%.Confident protein identifications required a minimum of two unique peptides with at least two corresponding spectra.Protein expression levels were quantified for each sample, utilizing the combined intensities of the three most prominent ion peaks from tryptic peptides, which enabled effective comparisons.Proteins and peptide features with a fold change ≥ 1.5 and a P-value < 0.05 between the WFB and oocyst wall groups were designated as differentially expressed proteins (DEPs) for further bioinformatics analysis.

Validation of proteomics data
To ensure the credibility of the TMT data, we performed Simple Western analysis using the WES ™ automated instrument (ProteinSimple, San Jose, CA, USA) in accordance with the manufacturer's protocols, to analyze the EnGAM22 and EnGAM59 protein expression profiles in the oocyst wall and WFBs.The proteins extracted from WFBs and the oocyst wall were diluted to a final concentration of 0.5 μg/μl with 5× Master Mix (ProteinSimple).Primary rabbit pAb against EnGAM22 and EnGAM59, prepared as described previously [15], were diluted 1:50 in Antibody Diluent II (ProteinSimple), and secondary antibodies were applied according to the manufacturer's instructions.Following automated separation and immunodetection, Compass software (Pro-teinSimple) was employed to visualize and analyze signal peaks, generating relative protein quantification from sample chromatograms and virtual gel images.

Detection of native protein EnPDI, EnTrx and EnPGK
The proteins of MZ-2, third-generation merozoites (MZ-3) (purified as previously reported [25]), GAM and unsporulated and sporulated oocysts (UO and SO) were abstracted using the method described previously [15], and their concentrations were determined as described above.Subsequently, 10 μg of total protein lysates was separated by 12% SDS-PAGE and transferred to nitrocellulose membranes (MilliporeSigma).The membranes were blocked with 3% BSA (Merck & Co., Rahway, NJ, USA) in PBS overnight at 4 °C, then incubated respectively with anti-rEnPDI, rEnTrx or rEnPGK mouse pAb (1:400 dilution) prepared by the method described previously [15].Following the incubation, the membranes were washed 3 times with 0.03% Tween-20/TBS (TBST) for 10 min each time and then probed with peroxidaseconjugated AffiniPure goat anti-mouse immunoglobulin G (IgG) (H+L; 1:10,000; Jackson ImmunoResearch Labs, West Grove, PA, USA) for 45 min at 37 °C.Finally, the membranes were developed in the presence of Highsig ECL Western blotting substrate (Tanon, Shanghai, China) after washing with TBST.Naïve sera from mice were used as a negative control.

Localization of EnPDI, EnTrx and EnPGK in GAM and UO
Indirect immunofluorescence analyses (IFA) were performed on tissue samples and purified parasites to investigate the localization of EnPDI, EnTrx and EnPGK proteins in E. necatrix according to the method reported in our previous study [15].Briefly, pathological tissue samples obtained from chickens sacrificed 156 h postinfection and from GAM and UO were immobilized on poly-L-lysine-coated glass slides and treated with 1.0% Triton X-100 for 20 min at room temperature.To quench endogenous peroxidase activity in the pathological tissue samples, the samples were incubated with 3% H 2 O 2 for 30 min, and antigen retrieval was accomplished by a 10-min exposure to 1 mg/ml trypsin (diluted with 0.1 M CaCl 2 ).Following fixation of both tissue samples and purified parasites in methanol (− 20 °C) and subsequent blocking  with 5% BSA, all samples were respectively incubated with anti-rEnPDI, rEnTrx or rEnPGK mouse pAb (1:100 dilution) at 37 °C for 1 h.Additionally, anti-rEnGAM22 rabbit pAb (1:100 dilution) and anti-rEnGAM59 rabbit pAb (1:100 dilution) were used as markers for WFB1 and WFB2 in the co-localization studies [15].After washing with 0.03% Tween 20/PBS (PBST), the samples were incubated with FITC-conjugated goat anti-rabbit IgG (1:100 dilution; MultiSciences, Hangzhou, China) and Cy3-conjugated goat anti-mouse IgG (1:100 dilution; Servicebio, Wuhan, China) in BSA/PBS at 37 °C for 1 h.Subsequently, the samples were visualized by laser (point) scanning confocal microscopy (LCM; Leica TCS SP8 STED microscope; Leica Microsystems GmbH, Wetzlar, Germany).Naïve sera from rabbits and mice served as negative controls.

Observation and SDS-PAGE analysis of the oocyst wall and WFBs
In our previous study, we demonstrated that anti-rEnGAM22 antibody specifically binds to WFB1s, while the anti-rEnGAM59 antibody specifically binds to WFB2s [15].In the present study, our double-immunofluorescence labeling analysis confirmed the presence of two types of WFBs in our isolations (Fig. 1a).Light microscopy revealed that the oocyst walls preparations had minimal contamination (Fig. 1b), and the BCA assay showed that the protein concentrations were similar in the three replicates of the oocyst wall preparations (5.316, 4.670 and 4.935 μg/μl, respectively) and the WFB preparations (3.096, 2.967 and 3.057 μg/μl, respectively).SDS-PAGE analysis showed that the protein bands mainly ranged from 11 to 135 kDa in the oocyst wall extracts and primarily from 35 to 135 kDa in the WFB extracts (Fig. 1c).These results confirmed that the samples were successfully prepared and could be used for further proteomics research.

Overview of primary data and protein identification
Of the 255,975 detected spectra, 232,083 were successfully identified, resulting in 28,449 peptide spectrum matches (PSMs) that encompassed 12,730 unique peptides.Editing and normalizing the data yielded a total of 3391 proteins having a confident prediction (FDR < 1%) across three biological replicates, of which 3059 proteins could be quantified using ≥ 2 peptides.Peptide length and count distribution revealed a predominant range of 8-13 amino acids (Fig. 2a), and > 80% of the identified proteins had a molecular size of < 200 kDa (Fig. 2b).As shown in Fig. 2c, a total of 2973 proteins were identified in the oocyst wall and 3009 proteins were identified in the WFBs, of which 36 were WFB-specific proteins (Additional file 1: Table S1).

Protein quantification and differential analysis
Applying a stringent cutoff (1.5-fold change), we identified a total of 1102 DEPs (P < 0.05) between WFBs and the oocyst wall (Fig. 3a; Additional file 2: Table S2).These DEPs included 506 upregulated and 596 downregulated proteins in the oocyst wall compared to the WFBs.The hierarchical clustering analysis of DEPs revealed highly similar and closely related expression patterns among the three biological replicates, effectively illustrating the distribution of DEPs and distinguishing the protein expression profiles between WFBs and oocyst wall (Fig. 3b).

Reliability analysis of proteomics data
The EnGAM22 (AHB64327.1)and EnGAM59 (AKN58547.1)proteins were selected for the validation of protein expression patterns identified by TMTbased quantitative proteomics.The proteomics results revealed downregulation of EnGAM22 in the oocyst wall, whereas there was no significant difference in EnGAM59 expression between the oocyst wall and WFBs.These expression patterns of EnGAM22 and EnGAM59 were confirmed by Simple Western analysis (Fig. 4; Additional file 3: Table S3), which suggested that our proteomic data was reliable.
The GO analysis of all the WFB proteins assigned 927, 1022 and 1370 proteins to the categories of biological processes (13 GO terms), cellular components (14 GO terms) and molecular function (12 GO terms), respectively (Fig. 5b).The top three GO terms were cellular process (29.65%), metabolic process (28.89%) and single-organism process (16.39%) in biological processes; cell (19.17%), cell part (18.06%) and membrane (17.66%) in the cellular components; and catalytic activity (44.58%), binding (42.53%) and structural molecule activity (5.44%) in molecular functions (Additional file 5: Table S5).A total of 1551 proteins were enriched in 1927 GO terms, and the top five enriched GO terms were metabolic process, intracellular, cell, organic substance metabolic process and primary metabolic process (Fig. 5c; Additional file 6: Table S6).Among the 36 WFB-specific proteins, six, two and 14 proteins were categorized into biological processes, cellular components and molecular functions, respectively (Additional file 4: Table S4).The GO annotation information suggests that these proteins may play pivotal roles in cellular metabolism, lipid synthesis, KEGG pathway annotations showed that, of all the WFB proteins, 1123 proteins were classified into five major categories and 34 subcategories (Fig. 5d; Additional file 7: Table S7).The category of translation accounted for the largest proportion at 12.30%, followed by folding, sorting and degradation (10.23%), and signal transduction (8.98%).KEGG pathway enrichment analysis revealed that the total proteins were mapped to 311 pathways, and the top five pathways included ribosome, spliceosome, RNA transport, glycolysis/gluconeogenesis and carbon metabolism.In addition, peroxisome, proteasome, protein processing in endoplasmic reticulum and fatty acid degradation were  S8).Among the 36 WFB-specific proteins, eight were annotated by KEGG (Additional file 4: Table S4), suggesting their potential crucial roles in various biological processes, including nucleotide metabolism, lipid synthesis, protein processing and spliceosome formation.
COG database annotations showed that, of all the WFB WFB proteins, 1193 proteins were classified into Fig. 4 Validation analysis of EnGAM22 and EnGAM59 by immunoblotting (Simple Western analysis).A1, B1 Protein expression levels of EnGAM22 and EnGAM59 in the WFBs and OW groups were analyzed using the capillary-based Simple Western automated system (ProteinSimple) and presented as virtual blots.A2, B2 Relative peak areas of EnGAM22 and EnGAM59 in the WFBs and OW groups were determined using the capillary-based Simple Western automated system.EnGAM22/EnGAM59, E. necatrix GAM protein 22/GAM protein 59; OW, oocyst wall; WFBs, wall-forming bodies 21 COG categories.The category exhibiting the greatest proportion was translation, ribosomal structure and biogenesis (20.29%), followed by posttranslational modification, protein turnover, chaperones (17.52%), general function prediction only (8.21%), energy production and conversion (7.54%), and signal transduction mechanisms (7.04%) (Fig. 5f; Additional file 9: Table S9).Among the 36 WFB-specific proteins, nine were annotated within COG categories (Additional file 4: Table S4), indicating the diverse roles of these proteins in biological processes such as cell signaling, metabolic pathways, cellular structure and post-transcriptional protein modification.

Bioinformatic analysis of DEPs between WFBs and the oocyst wall
In the GO analysis, 235, 198 and 290 DEPs were assigned to the categories of biological processes (12 GO terms), cellular components (9 GO terms) and molecular function (8 GO terms), respectively (Fig. 6a; Additional file 10: Table S10).The top three GO terms were similar with these of WFB proteins, expect for the replacement of 'membrane' with 'macromolecular complex' in cellular components.The 833 DEPs were enriched in 1191 GO terms, and the top five enriched GO terms were disulfide oxidoreductase activity, oxidoreductase activity, protein disulfide oxidoreductase activity, coenzyme biosynthetic process and electron carrier activity (Fig. 6b; Additional file 11: Table S11).
KEGG pathway annotations showed that 394 DEPs were classified into five major categories and 31 subcategories (Fig. 6c; Additional file 12: Table S12).The category of translation accounted for 13.71% DEPs, followed by global and overview maps (9.64%) and carbohydrate metabolism (9.39%).KEGG pathway enrichment analysis revealed that all DEPs were mapped to 166 pathways and that the top five pathways included RNA transport, fatty acid biosynthesis, fatty acid metabolism, peroxisome and protein processing in the endoplasmic reticulum.Fatty acid degradation, ubiquinone and other terpenoid-quinone biosynthesis and ubiquitin-mediated proteolysis were also ranked in the top 30 pathways (Fig. 6d; Additional file 13: Table S13).
A total of 391 DEPs were classified into 21 COG categories, of which the top five categories were similar with these of WFB proteins, expect that 'signal transduction mechanisms' was replaced with 'carbohydrate transport and metabolism.' The categories 'translation, ribosomal structure and biogenesis' (20.74%) and 'posttranslational modification, protein turnover, chaperones' (20.72%) were also ranked in the top two among 21 COG categories (Fig. 6e; Additional file 14: Table S14).

Analysis and identification of proteins potentially involved in oocyst wall formation
The results of the GO, KEGG and COG analyses suggest that 108 of 3059 quantified proteins are involved in oocyst wall formation.Based on their possible functions, these proteins could be classified into six distinct groups (Fig. 7; Table 2), including compositional proteins of the oocyst wall (15%), protease (14%), oxidoreductase (26%), proteins involved in glycosylation (8%), proteins involved in synthesis of the acid-fast lipid layer (23%) and proteins related to transport (14%).In addition, a total of 20 glycolytic enzymes were identified from WFBs and the oocyst wall (Additional file 15; Table S15).Subsequently, EnPDI, EnTrx and EnPGK were selected for further study.

Cloning and expression of EnPDI, EnTrx and EnPGK
The complementary DNA (cDNA) sequence of EnPDI protein was amplified to a length of 1473 bp (GenBank accession number: OR105511; Additional file 16: Figure S1A1) and encoded a 490-amino acid (aa) polypeptide with a 21-aa signal peptide and a predicted molecular weight of approximately 53.96 kDa; the deduced protein sequence (GenBank: WMD29389.1)was 98.6% sequence identity to the sequence deposited in the NCBI database (XP_013435909.1)(Additional file 17: Figure S2A).The cDNA sequence of EnTrx protein was amplified to a length of 1329 bp (GenBank accession number: OR105512; Additional file 16: Figure S1A2) and encoded a 442-aa polypeptide with a 38-aa signal peptide and a predicted molecular weight of approximately 49.57 kDa; the deduced protein sequence (GenBank: WMD29390.1)was 100.0%sequence identity to the sequence deposited in the NCBI database (XP_013439420.1)(Additional file 17: Figure S2B).The cDNA sequence of EnPGK protein was amplified to a length of 1206 bp (GenBank Accession number: OR105509, Additional file 16: Figure S1A3) and encoded a 401-aa polypeptide without a signal peptide and with a predicted molecular weight of approximately 42.33 kDa; the deduced protein sequence (GenBank: WMD29387.1)was 90.5% sequence identity to the sequence deposited in the NCBI database (XP_013438037.1)(Additional file 17: Figure S2C).
The recombinant bacteria containing expression vectors (Additional file 16: Figure S1B1-B3) were induced with IPTG.rEnPDI was expressed in soluble form, with a molecular weight of about 57 kDa, while rEnTrx and rEnPGK were expressed as inclusion bodies, with a molecular weight of about 52 and 45 kDa, respectively.The bands of the expected size (57, 52 and 45 kDa, respectively) could be detected when bacterial lysates containing the recombinant protein were probed with the anti-6×His tag monoclonal antibody (Fig. 8a1-a3).
No reaction was detected when negative control serum was used (Additional file 18: Figure S3; Additional file 19: Figure S4)..

Discussion
Based on the results of this study: (i) two tyrosine-rich precursor proteins (gam56 and gam82) were proteolytically processed into smaller peptides that were incorporated into the developing oocyst wall of E. maxima; (ii) oocysts gave a characteristic blue autofluorescence at 340-360 nm; (iii) dityrosine and 3,4-dihydroxyphenylalanine (DOPA) were biochemically detectable in oocyst extracts; and (iv) peroxidase activity was detectable in the WFBs of macrogametes.Belli et al. proposed a model for the wall formation of coccidian oocyst [5]: large tyrosine-rich proteins or scaffolding proproteins are synthesized and stockpiled in WFBs of macrogametes, and at a certain developmental time point, the proproteins are processed to smaller, tyrosine-rich proteins; the tyrosine residues in the proteins undergo oxidative phenolic coupling by an enzyme, such as peroxidase, which leads to the formation of protein-dityrosine, dehydration and oocyst wall hardening.Belli et al. also suggested that the molecular machinery involved in the assembly of the oocyst wall, including precursor proteins, processing enzymes, cross-linking enzymes and cofactors, were housed in the WFBs in macrogametocytes [5].However, the exact contents of WFBs remain unclear to date.In the present study, we applied TMT labeling coupled with LC-MS/MS to profile and compare the proteomes of E. necatrix WFBs and the oocyst wall.A total of 3009 proteins were identified in the WFBs, with 108 identified proteins proposed to be involved in oocyst wall formation.Based on their possible functions, these proteins were classified into six distinct groups: compositional proteins of the oocyst wall, protease, oxidoreductase, proteins involved in glycosylation, proteins involved in synthesis of the acid-fast lipid layer and proteins related to transport.To the best of our knowledge, this is the first report on the proteomes of WFBs and the oocyst wall of E. necatrix.

Compositional proteins of the oocyst wall
Three groups of gametocyte proteins have been confirmed to participate in oocyst wall formation.One group consists of tyrosine-rich glycoproteins that localize to WFB2 of macrogametocytes and the inner wall of Eimeria oocysts, including Gam82, Gam56 and Gam59 [8,26].Another group is histidine-proline-rich protein that localizes to WFB1 of macrogametocytes and the outer wall of Eimeria oocyst, with Gam22 being a prominent representative of this group [15].The third group is cysteine-rich proteins (namely oocyst wall protein [OWP]) that localize to WFB1 and the inner wall of Cryptosporidium oocyst [27], and to WFB1 and the outer wall of Eimeria nieschulzi oocyst [28].In the present study, Gam82 (XP_013441002.1,AAO47083.2),Gam56 (XP_013333565.1,XP_013333564.1,XP_013441001.1,XP_013232286.1),Gam59 (AKN58547.1),Gam22 (AHB64327.1)and OWP (XP_013440134.1) were identified in both the WFBs and the oocyst wall of E. necatrix.Of these proteins, Gam56, Gam59 and Gam22 have been identified and found to be involved in formation of the inner and outer wall of the E. necatrix oocyst [15,26], whereas the functions of Gam82 and OWP need to be confirmed.
Ferguson et al. found that the microneme protein MIC4 or an MIC4-like protein was expressed within the WFB1a of the macrogamete and associated with oocyst wall formation in T. gondii [29].Proteomic analysis of fractionated Toxoplasma oocysts revealed the distinct abundance of PAN domain-containing proteins that are characterized by a disulfide bridge folding pattern [19].The transcripts for PAN domain-containing proteins also exhibited upregulation in E. tenella gametocytes [30].In the present study, four MIC proteins (XP_013440596.1, XP_013438712.1,XP_013433151.1,XP_013231935.1)and three PAN domain-containing proteins (XP_013235137.1, XP_013432524.1,XP_013434151.1)were identified.However, further study is required to determine the precise roles of these proteins in oocyst wall formation.
PDI and Trxs belong to the thioredoxin superfamily, whose members share a common structural motif named the thioredoxin fold.They are involved in disulfide oxidoreduction and/or isomerization [11].Previous studies revealed that Cryptosporidium oocyst wall proteins (COWPs) and their orthologs in T. gondii participate in the wall formation of oocysts [36,37], and that these cysteine motif-containing proteins are cross-linked via disulfide bonds between the cysteine residues [27,36].A recent study showed that EnOWP13 is a protein specifically localized to WFBI and a component of the outer oocyst wall of E. nieschulzi, with the results suggesting the existence of isopeptide bonds in the oocyst wall that were regularly stimulated by transglutaminase activity [28].In the present study, one oocyst wall protein (OWP), four PDIs and nine Trxs were detected, of which EnPDI (XP_013435909.1) and EnTrx (XP_013439420.1) were chosen for further study.The native EnPDI protein (approx.55 kDa) was detected in MZ-2, MZ-3, GAM and SO, and was localized in WFB1s and WFB2s.The native EnTrx protein (approx.50 kDa) was present in MZ-2, MZ-3, GAM and UO, and was localized in WFB2s rather than WFB1s.However, both EnPDI and EnTrx were localized in the outer layer of the oocyst wall.These results implied that apart from via dityrosine bonds catalyzed by peroxidase, the oocyst wall proteins of Eimeria (such as OWP) may undergo cross-linking via isopeptide bonds or disulfide bonds catalyzed by PDIs or Trxs, ultimately contributing to the rigidification of the oocyst wall.The precise roles of EnPDI and EnTrx in oocyst wall formation still has to be revealed, and whether the OWP (XP_013440134.1)participates in oocyst wall formation of E. necatrix is a question for future research.

Proteins involved in synthesis of the acid-fast lipid layer
A previous study showed that the abundant neutral lipids such as cholesterol and triglycerides in WFB1, which align at the periphery of gametocytes and release their contents as "rafts", contribute to the formation of the patchwork pattern in the outer oocyst wall [39].The acid-fast stains, which typically adhere to lipids in mycobacterial cell walls, have also been observed on the oocyst wall [40].These findings suggested that coccidia build a waxy coat of acid-fast lipids in the oocyst wall that makes them resistant to environmental stress [40].In the present study, we identified 25 proteins that might be involved in synthesizing the acid-fast lipid layer, including one very long-chain acyl-CoA synthetase (XP_013439980.1),two type I fatty acid synthases (XP_013433087.1,KFG59574.1),two sterol O-acyltransferases (XP_013438000.1,XP_013233477.1),two polyketide synthases (XP_013439400.1,XP_013434943.1),two phospholipase/carboxylesterases (XP_013440740.1,XP_013228414.1),three peroxisomal multifunctional enzymes (XP_013228107.1,XP_013228145.1,XP_013432721.1)and four glycerol-3-phosphate dehydrogenases (XP_013439788.1,XP_013438016.1,XP_013440415.1,XP_013434136.1).Similarly, the transcripts encoding these proteins associated with acid-fast lipid layer synthesis have been found to be upregulated in the gametocytes of E. tenella and E. necatrix [30,31].However, the precise roles of these proteins in the development of the lipid layer still needs to be determined.

Glycolytic enzymes identified from WFBs and oocyst wall
Interestingly, a total of 20 glycolytic enzymes were identified from WFBs and the oocyst wall, of which the PGK (XP_013438037.1)exhibited higher levels of expression in the WFBs than in the oocyst wall of E. necatrix.Further research revealed that the EnPGK is present in MZ-2, MZ-3 and GAM, and that it is localized in WFB1s and the outer layer of the oocyst wall but not in WFB2s.A previous study confirmed that PGK is a bona fide cell wall protein of C. albicans [12].In S. mansoni, the subcellular localization of glycolytic enzymes directly beneath the tegument surface suggests the potential involvement of these enzymes in utilizing incoming glucose for ATP production, which is crucial for various physiological processes, such as nutrient and solute uptake, as well as cytoskeletal rearrangements [13].How these glycolytic enzymes participate in the formation of oocyst wall still needs to be elucidated.

Conclusions
In summary, our research represents the first attempt to investigate the protein abundance in WFBs and the oocyst wall of E. necatrix using TMT peptide labeling coupled with the LC-MS/MS quantitative proteomics technique.A total of 3009 and 2973 proteins were identified from WFBs and the oocyst wall of E. necatrix, respectively.A total of 108 proteins were proposed to be involved in oocyst wall formation, including compositional proteins of the oocyst wall, protease, oxidoreductase, proteins involved in glycosylation, proteins involved in the synthesis of the acid-fast lipid layer and proteins related to transport.Furthermore, we confirmed that EnPDI, EnTrx and EnPGK participated in the formation of the oocyst wall.While further functional studies are needed to fully elucidate the roles of these proteins in the formation of the oocyst wall, our results provide new insights into the molecular mechanisms underlying the formation of oocyst wall of Eimeria parasites.Moreover, our work can help in the development of novel therapeutic agents and vaccines aimed at combating coccidian transmission.

b
Primers for prokaryotic expression c The protective bases are shown in italics and the restriction sites are underlined Primer name Primer sequence 5ʹto 3ʹ c EnPDI-F a ATG AAA AGA CCT TTC TTG CTCGG EnPDI-R a TCA GAG TTC CTC GCC CTT GTC EnPDI-pET28aF b CGGGA TCC GCA GCA GCA GCA GCA GAG AACAA EnPDI-pET28aR b CCG CTC GAG TCA GAG TTC CTC GCC CTT GTCGG EnTrx-F a ATG GCT TTG GGC GTT GGC TTA GGA A EnTrx-R a CTA AAG CTC TTC TTT CTT TGTGT EnTrx-pET28aF b CGGGA TCC GCG CGG CCG CTG ATC GAC CCCAT EnTrx-pET28aR b CGGAA TTC CTA AAG CTC TTC TTT CTT TGTGT EnPGK-F a ATG CGC GTG GAC TTC AAC GTGCC EnPGK-R a TCA CTT TGA AGA CAA AGC TGC CAC TC EnPGK-pET28aF b CGGGA TCC ATG CGC GTG GAC TTC AAC GTGCC EnPGK-pET28aR b ATA GTT TAGCG GCC GCTCA CTT TGA AGA CAA AGC TG

Fig. 2 Fig. 3
Fig. 2 Overview of tandem mass tag-based quantitative proteomics for primary data and protein identification.a Distribution of peptide fragment lengths derived from WFBs and OW.b Distribution of identified proteins with different molecular weights.c Venn diagram showing overlapping proteins between WFBs and OW.OW, Oocyst wall; WFBs, wall-forming bodies

Fig. 5
Fig. 5 Bioinformatic analysis results of WFB proteins.a WFB protein functional annotation results from GO, KEGG and COG databases.Each circle in the diagram represents the annotation result of a database, the overlapping portion represents the protein annotated jointly by multiple databases and the nonoverlapping portion represents the protein annotated separately by the corresponding database.b Summary of second-level GO assignments for WFB proteins.The x-axis represents the second-level functional categories from the GO database, and the y-axis represents the number of WFBs protein in each second-level functional category.c Top 30 GO enrichment results for WFB proteins.The x-axis represents the results of Input frequency/Background frequency in the enrichment analysis, and the y-axis represents enriched GO terms.d Summary of second-level KEGG pathway analysis for WFB proteins.The x-axis represents the number of genes in each pathway, and the y-axis indicates the main second-level pathways.e Top 30 KEGG enrichment results for WFB proteins.The x-axis represents the results of Input frequency/ Background frequency in the enrichment analysis, and the y-axis represents enriched KEGG pathway terms.f COG function classification of WFB proteins.The x-axis indicates the different categories of COG, and the y-axis indicates the frequency of different categories of COG.COG, Cluster of Orthologous Groups database; GO, Gene Ontology resource; KEGG, Kyoto Encyclopedia of Genes and Genomes database; WFBs, wall-forming bodies

Fig. 6
Fig. 6 Bioinformatic analysis results of DEPs between WFBs and the OW. a Summary of second-level GO assignments for DEPs.The x-axis represents the second-level functional categories from the GO database, and the y-axis represents the percentage (left) and number (right) of DEPs corresponding to the genes in each second-level functional category.b Top 30 GO enrichment results for DEPs.The x-axis represents the results of Input frequency/Background frequency in the enrichment analysis, and the y-axis represents enriched GO terms.c Summary of second-level KEGG pathway analysis for DEPs.The x-axis represents the number of genes in each pathway, and the y-axis indicates the main second-level pathways.d Top 30 KEGG enrichment results for DEPs.The x-axis represents the results of Input frequency/Background frequency in the enrichment analysis, and the y-axis represents enriched KEGG pathway terms.e COG function classification of DEPs.The x-axis indicates the different categories of COG, and the y-axis indicates the frequency of different categories of COG.COG, Cluster of Orthologous Groups database; DEPs, differentially expressed proteins; GO, Gene Ontology resource; KEGG, Kyoto Encyclopedia of Genes and Genomes database; OW, oocyst wall; WFBs, wall-forming bodies

Fig. 7
Fig. 7 Functional classification of 108 proteins potentially involved in oocyst wall formation.Biological functions of these proteins were manually assigned based on GO, KEGG, COG, or previously published annotations.The accompanying text illustrates the number and proportion of proteins characterized by their biological functions.COG, Cluster of Orthologous Groups database; GO, Gene Ontology resource; KEGG, Kyoto Encyclopedia of Genes and Genomes database; OW, outer oocyst wall; IW, inner oocyst wall; WFB1/2, wall-forming bodies type 1/type 2

Table 1
Primer sequence En Eimeria necatrix, F forward primer, PDI protein disulfide isomerase, pET28a bacterial vector, PGK phosphoglycerate kinase, R reverse primer, Trx thioredoxin a Primers for complete gene

Table 2
Classification of proteins involved in oocyst wall formation